RESUMEN
Neuromuscular junction (NMJ) disruption is an early pathogenic event in amyotrophic lateral sclerosis (ALS). Yet, direct links between NMJ pathways and ALS-associated genes such as FUS, whose heterozygous mutations cause aggressive forms of ALS, remain elusive. In a knock-in Fus-ALS mouse model, we identified postsynaptic NMJ defects in newborn homozygous mutants that were attributable to mutant FUS toxicity in skeletal muscle. Adult heterozygous knock-in mice displayed smaller neuromuscular endplates that denervated before motor neuron loss, which is consistent with 'dying-back' neuronopathy. FUS was enriched in subsynaptic myonuclei, and this innervation-dependent enrichment was distorted in FUS-ALS. Mechanistically, FUS collaborates with the ETS transcription factor ERM to stimulate transcription of acetylcholine receptor genes. Co-cultures of induced pluripotent stem cell-derived motor neurons and myotubes from patients with FUS-ALS revealed endplate maturation defects due to intrinsic FUS toxicity in both motor neurons and myotubes. Thus, FUS regulates acetylcholine receptor gene expression in subsynaptic myonuclei, and muscle-intrinsic toxicity of ALS mutant FUS may contribute to dying-back motor neuronopathy.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Regulación de la Expresión Génica/fisiología , Degeneración Nerviosa/fisiopatología , Unión Neuromuscular/metabolismo , Proteína FUS de Unión a ARN/fisiología , Adulto , Esclerosis Amiotrófica Lateral/patología , Animales , Células Cultivadas , Femenino , Técnicas de Sustitución del Gen , Humanos , Masculino , Ratones , Ratones Noqueados , Neuronas Motoras/patología , Fibras Musculares Esqueléticas/patología , Unión Neuromuscular/patología , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Receptores Colinérgicos/metabolismo , Adulto JovenRESUMEN
Excitotoxicity driven by overactivation of NMDA receptors represents a major mechanism of acute and chronic neurological and neurodegenerative disorders. Negative allosteric modulators interacting with the ifenprodil binding site of the NMDA receptor are able to interrupt this ongoing neurodamaging process. Starting from the potent 3-benzazepine-1,7-diol 4a novel NMDA receptor antagonists were designed by modification of the N-(4-phenylbutyl) side chain. With respect to developing novel fluorinated PET tracers, regioisomeric fluoroethoxy derivatives 11, 12, 14, and 15 were synthesized. Analogs 19 and 20 with various heteroaryl moieties at the end of the N-side chain were prepared by Sonogashira reaction and nucleophilic substitution. The fluoroethyl triazole 37 was obtained by 1,3-dipolar cycloaddition. In several new ligands, the flexibility of the (hetero)arylbutyl side chain was restricted by incorporation of a triple bond. The affinity towards the ifenprodil binding site was tested in an established competition assay using [3H]ifenprodil as radioligand. Introduction of a fluoroethoxy moiety at the terminal phenyl ring, replacement of the terminal phenyl ring by a heteroaryl ring and incorporation of a triple bond into the butyl spacer led to considerable reduction of GluN2B affinity. The phenol 15 (Kiâ¯=â¯193â¯nM) bearing a p-fluoroethoxy moiety at the terminal phenyl ring represents the most promising GluN2B ligand of this series of compounds. With exception of 15 showing moderate σ2 affinity (Kiâ¯=â¯79â¯nM), the interaction of synthesized 3-benzazepines towards the PCP binding site of the NMDA receptor, σ1 and σ2 receptors was rather low (Kiâ¯>â¯100â¯nM).
Asunto(s)
Benzazepinas/química , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sitios de Unión , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Cabeza (caz) is the single Drosophila melanogaster orthologue of the human FET proteins FUS, TAF15, and EWSR1, which have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In this study, we identified Xrp1, a nuclear chromatin-binding protein, as a key modifier of caz mutant phenotypes. Xrp1 expression was strongly up-regulated in caz mutants, and Xrp1 heterozygosity rescued their motor defects and life span. Interestingly, selective neuronal Xrp1 knockdown was sufficient to rescue, and neuronal Xrp1 overexpression phenocopied caz mutant phenotypes. The caz/Xrp1 genetic interaction depended on the functionality of the AT-hook DNA-binding domain in Xrp1, and the majority of Xrp1-interacting proteins are involved in gene expression regulation. Consistently, caz mutants displayed gene expression dysregulation, which was mitigated by Xrp1 heterozygosity. Finally, Xrp1 knockdown substantially rescued the motor deficits and life span of flies expressing ALS mutant FUS in motor neurons, implicating gene expression dysregulation in ALS-FUS pathogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Neuronas Motoras/metabolismo , Mutación , Proteínas de Unión al ARN/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Técnicas de Silenciamiento del Gen , Humanos , Dominios Proteicos , Proteínas de Unión al ARN/genética , Factor de Transcripción TFIID/genéticaRESUMEN
Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, we assessed the requirements for translation initiation factors during dendrite pruning. We found that the canonical cap-binding complex eIF4F is dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. We propose that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Ecdisona/genética , Factor 4E Eucariótico de Iniciación/genética , Animales , Diferenciación CelularRESUMEN
Motor neuron-extrinsic mechanisms have been shown to participate in the pathogenesis of ALS-SOD1, one familial form of amyotrophic lateral sclerosis (ALS). It remains unclear whether such mechanisms contribute to other familial forms, such as TDP-43 and FUS-associated ALS. Here, we characterize a single-copy mouse model of ALS-FUS that conditionally expresses a disease-relevant truncating FUS mutant from the endogenous murine Fus gene. We show that these mice, but not mice heterozygous for a Fus null allele, develop similar pathology as ALS-FUS patients and a mild motor neuron phenotype. Most importantly, CRE-mediated rescue of the Fus mutation within motor neurons prevented degeneration of motor neuron cell bodies, but only delayed appearance of motor symptoms. Indeed, we observed downregulation of multiple myelin-related genes, and increased numbers of oligodendrocytes in the spinal cord supporting their contribution to behavioral deficits. In all, we show that mutant FUS triggers toxic events in both motor neurons and neighboring cells to elicit motor neuron disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Neuronas Motoras/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Axones/metabolismo , Axones/patología , Citoplasma/metabolismo , Citoplasma/patología , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiología , Neuronas Motoras/patología , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Oligodendroglía/metabolismo , Oligodendroglía/patología , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/genética , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.
Asunto(s)
Neuronas Motoras/metabolismo , Proteína FUS de Unión a ARN/genética , Animales , Encéfalo/metabolismo , Citoplasma/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Proteína FUS de Unión a ARN/metabolismo , Médula Espinal/metabolismoRESUMEN
Dominant mutations in five tRNA synthetases cause Charcot-Marie-Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA(Gly) aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Glicina-ARNt Ligasa/genética , Neuronas Motoras/metabolismo , Movimiento , Biosíntesis de Proteínas/genética , Células Receptoras Sensoriales/metabolismo , Tirosina-ARNt Ligasa/genética , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila , Humanos , Esperanza de Vida , Neuronas Motoras/patología , Mutagénesis Sitio-Dirigida , Mutación , Unión Neuromuscular/patología , Fenotipo , Células Receptoras Sensoriales/patologíaRESUMEN
To expand the rich genetic toolkit of Drosophila melanogaster, we evaluated whether introducing FRT or LoxP sites in endogenous genes could allow for cell-type-specific gene inactivation in both dividing and postmitotic cells by GAL4-driven expression of FLP or Cre recombinase. For proof of principle, conditional alleles were generated for cabeza (caz), the Drosophila homolog of human FUS, a gene implicated in the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Upon selective expression in neurons or muscle, both FLP and Cre mediated caz inactivation in all neurons or muscle cells, respectively. Neuron-selective caz inactivation resulted in failure of pharate adult flies to eclose from the pupal case, and adult escapers displayed motor performance defects and reduced life span. Due to Cre-toxicity, FLP/FRT is the preferred system for cell-type-specific gene inactivation, and this strategy outperforms RNAi-mediated knock-down. Furthermore, the GAL80 target system allowed for temporal control over gene inactivation, as induction of FLP expression from the adult stage onwards still inactivated caz in >99% of neurons. Remarkably, selective caz inactivation in adult neurons did not affect motor performance and life span, indicating that neuronal caz is required during development, but not for maintenance of adult neuronal function.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Silenciador del Gen , Neuronas/metabolismo , Alelos , Animales , Animales Modificados Genéticamente , Técnicas de Inactivación de Genes , Marcación de Gen , Recombinación Homóloga , Actividad Motora/genética , Neuronas Motoras/metabolismo , Músculos/metabolismo , Especificidad de Órganos/genéticaRESUMEN
Na última década, houve um aumento significativo na utilização de resinas compostas para restaurações em dentes posteriores. A preservação do tecido dental sadio e a semelhança deste material com a estrutura dental, talvez sejam os principais responsáveis para a ocorrência deste fato. Além disso, com a aplicação crescente de métodos de prevenção e melhora dos hábitos de higiene bucal, houve uma diminuição na incidência de cárie dental, que associada aos avanços tecnológicos de materiais, equipamentos e técnicas restauradoras, possibilitou a realização de restaurações mais conservadoras através de materiais estéticos. O objetivo deste trabalho é através da apresentação de um caso clínico, demonstrar técnicas de execução de restaurações conservativas em dentes posteriores com resina composta